Fig. 1: KMT2A::AFF1 impacts proliferation and lineage output of fetal HSPCs.
A Schematic illustration of the model. Human KMT2A::AFF1 was placed under the control of a Tet-OP promotor; the tissue recombinase (tTA) had a STOP cassette flanked by LoxP sites. Strain specific Cre-recombinase was used to induce expression of KMT2A::AFF1. B Relative expression (normalized to B-actin) of KMT2A::AFF1 in purified HSCs, LMPPs and ProBs from E18.5 KMT2A::AFF1Vav-Cre+ fetal livers (FLs). Box plots define lower and upper quartiles, and whiskers min to max values (4 FACS experiments). C, D Frequencies of LSK, HSCs and LMPPs as percent of CD45+ cells in control and KMT2A::AFF1Vav-Cre+ FLs at (C) E14.5 (3 experiments) and (D) E18.5 (6 experiments). E Flow cytometry plots of LSK compartment in control and KMT2A::AFF1Vav-Cre+ FLs at E18.5. Cells were gated CD45+Lin–CD19–. Further gating is indicated in the figure showing LSK (top) and LMPPs (bottom). Numbers are mean percentages of total CD45+ cells. F Representative photos of E18.5 HSCs (left) and LMPPs (right) bulk liquid cultures at day 7. Genotypes (control and KMT2A::AFF1Vav-Cre+) are indicated. G Schematic illustration of single-cell OP9 co-culture workflow and subsequent evaluation of B and myeloid linage potentials using flow cytometry. H, I Single HSCs (H) and LMPPs (I) were co-cultured on OP9 stroma. Frequency of colonies (left) and linage output (right) for control and KMT2A::AFF1Vav-Cre+ FLs at E18.5 are shown. Colonies were defined as B, myeloid; B and myeloid (B & My) or not designated to any of these (none) (3 experiments). J Cell cycle analysis of HSCs (left) and MPPs (LSKCD150-CD48+)(right) at E18.5. Mean percentages of cells in G0/G1 and S/G2/M for each population are shown (1–2 embryos per genotype,1 experiment). Bars show means ± SD, and each dot represents an individual embryo. *p ≤ 0.05; ** p ≤ 0.01; ****p ≤ 0.0001; n.s. not significant.
